C. crescentus is a gram-negative microorganism that divides unequally to produce a nonmotile stalked cell and a smaller, flagellated swarmer cell, each with different developmental sequences. This organism is an ideal model system for the study of stem cell differentiation. Two related aspects of development in C. crescentus are of interest. (1) Temporal patterns - How is the program for an ordered series of structural and functional changes encoded in a cell? and (2) Spatial patterns - How is structure and functional information localized within a cell in a way to lead to unequal cell division? Our three main approaches are: A. Spatial Localization - Swarmer and stalked cells are being compared biochemically and genetically to identify differences responsible for their characteristic developmental cycles; conditional mutants that effect the normal development from swarmer to stalked cell will be isolated and analyzed for the defects responsible. B. Sequencing of Developmental Events - To determine how steps in a developmental pathway are ordered, we are determining whether individual steps, e.g. septation, motility, stalk formation and DNA synthesis, are dependent or independent of a previous step. Some steps in C. crescentus are on dependent pathways (unpublished), and it is proposed to look at specific proteins in these pathways to determine the mechanism of this regulation. C. Control of Gene Expression - Related to questions of ordering developmental events are studies of transcriptional specificity. We know that RNA synthesis is needed for most events to occur normally in the cell cycle. By using DNA-RNA hybridization techniques, it is planned to determine what parts of the genome are expressed at various stages of the cell cycle and to design assays for RNA species that are stage specific.